The “.txt” Sequence Listing filed with this application by EFS and which is entitled 54887_0006_ST25.txt, is 7 kilobytes in size and which was created on Jan. 30, 2012 is hereby incorporated by reference.
Throughout this application various publications are referred to in brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and of all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Breast cancer is one of the most common malignant diseases in the United States: 1 in 8 women are diagnosed with breast cancer during their lifetime (NIH website, breast cancer statistics). The main cause of death for breast cancer patients arises from dissemination of the primary tumor by metastases to other organs, a process that may only manifest as long as 10 or more years after initial diagnosis [1].
Currently established clinical prognostic criteria, including the histopathologic grade of the tumor, tumor size, the presence of the lymph node metastasis and hormone receptor status, can predict systemic metastatic potential in only a subgroup of patients with breast cancer. Microarray gene expression platforms, such as the MammaPrint™ 70 gene signature, are emerging as predictors of distant metastasis [1,2] but lack broad applicability [1] and offer relatively limited predictive power [3]. Therefore, novel prognostic markers are needed to identify patients with the high risk of developing metastasis to drive clinical treatment decisions.
About 90% of human malignancies are carcinomas, tumors of epithelial origin [4]. The early steps in carcinoma metastasis often bear a striking resemblance to developmental programs involving Epithelial-to-Mesenchymal Transition (EMT), a process that converts polarized organized epithelial cells into isolated, migratory cells with a mesenchymal morphology [5]. A growing body of work implicates EMT-like mechanisms in tumor cell invasion and dissemination in experimental systems, and recently, in human cancer [6,7]. Normal epithelia are comprised of cells with aligned apical-basal polarity that are interconnected laterally by several types of junctions including adherens junctions (AJs), which play important roles in establishing and regulating cell-cell adhesion [8]. E-cadherin, the major component of epithelial AJs, is a homophilic transmembrane protein that engages E-cadherin molecules on neighboring cells, and loss of functional E-cadherin is a hallmark of EMT. During EMT, apico-basolateral polarity is lost, cell-cell junctions dissolve and the actin cytoskeleton is remodeled to endow cells with mesenchymal characteristics including an elongated, migratory and invasive phenotype.
Importantly, as a consequence of EMT, cells may escape the tumor, invade the surrounding tissue and migrate towards blood vessels or lymphatic vessels guided by the cells and extracellular matrix present in their microenvironment [9]. Thus, EMT, a mechanism important for embryonic development, plays a critical role during malignant transformation.
While much is known regarding the regulation of EMT at the transcriptional level, alternative splicing of several genes has also been correlated with EMT progression. The extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been extensively investigated.
The molecular mechanisms underlying EMT have been studied extensively in the last decade. EMT-inducing growth factors can trigger signaling cascades that activate a network of transcription factors, such as ZEB-1, Goosecoid, FOXC2 and Twist [17], that orchestrate the EMT program; ectopic expression of a number of the EMT-associated transcription factors can initiate the program as well. Twist, a potent EMT driver, was identified originally as an inducer of mesoderm formation in Drosophila [18]. Ectopic Twist expression in epithelial cells results in loss of E-cadherin-mediated cell-cell adhesion, acquisition of mesenchymal markers and increased motility of isolated cells [19], a hallmark of the mesenchymal phenotype. E-cadherin expression is suppressed by several EMT-inducing transcription factors [20,21], while some mesenchymal markers are activated directly by this same repertoire of factors.
The control of EMT is likely also subject to regulation at post-transcriptional levels such as alternative pre-mRNA splicing. Alternative splicing expands the diversity of the proteome by producing multiple mRNA isoforms from each gene [22]. More than 90% of human genes are estimated to undergo alternative splicing, with a majority of alternative splicing events exhibiting tissue-specific splicing differences [23]. A variety of cancer-associated genes express alternatively spliced isoforms [24], indicating that regulation at the level of splicing may play important roles in cancer onset and progression. Alternative splicing of FGFR2 correlates with EMT in rat bladder carcinoma cells, where mutually exclusive inclusion of one of two exons defines the ligand binding specificity of the receptor during EMT [25]. ENAH (also known as Mena), an actin cytoskeleton regulatory protein, contains a small coding exon 11a that is included exclusively within epithelial cells and is excluded in mesenchymal cell lines and during EMT [26,27]. Alternative splicing of p120catenin (CTNND1) generates protein isoforms that display opposite effects on cell motility in epithelial and mesenchymal cells [28].
Recently, two epithelial-specific RNA binding proteins, ESRP1 and ESRP2, homologs of the nematode splicing factor Sym-2, were identified in a screen for FGFR2 splicing regulators [27]. REFOX2 (formerly “Fox2”) splicing factor has been recently demonstrated to regulate subtype-specific splicing in a panel of breast cancer cell lines [29]. The ESRPs and RBFOX2 promote epithelial splicing of a number of transcripts including FGFR2 and ENAH, some of which play important roles in EMT [27,30]. Loss of ESRPs in epithelial cells promotes EMT-like changes in cell morphology [31]. However, the full extent of alternative splicing during EMT and its functional consequences to cell phenotype has yet to be elucidated.
The present invention has identified signatures of multi-exon genes that undergo alternative splicing during EMT and are predictive of metastasis.